In this renewal application, we propose to use a novel cellular magnetic resonance imaging (MRI) technology to visualize the trafficking of tumor infiltrating lymphocytes (TILs) in head and neck cancer (HNC) patients. In the first funding cycle, we successfully developed and implemented ?first-into-man? clinical translation of a disruptive imaging technology based on a novel perfluorocarbon (PFC) emulsion probe (CS-1000) that employs fluorine- 19 (19F) MRI cell detection. Overall, cell populations of interest are intracellularly-labeled in culture using non- toxic PFC as an additive to the culture media. Following transfer to the subject, cells are tracked in vivo using 19F MRI. The fluorine inside the cells yields cell-specific images, with no background signal. Images are quantified to measure apparent cell numbers at sites of accumulation. In the pilot trial, immunotherapeutic dendritic cells (DCs) were labeled with CS-1000 and longitudinally detected in colorectal cancer patients using 19F MRI. Building on these efforts, we aim to use this technology for imaging TILs in HNC. HNC is the sixth most common cancer worldwide. In the United States, HNC accounts for 3% of all cancers diagnosed annually and 2% of cancer- related deaths, with poor prognosis (<2 mo survival) after reoccurrence. Immunotherapy is emerging as a key anti-cancer strategy with the potential to provide patient-specific, less toxic and more efficacious treatments. TILs have proven successful in melanoma, and a major effort is underway at UCSD to develop this therapy for HNC. Although TIL therapies have been used in hundreds of patients to date, fundamental questions remain about tumor homing and cell survival of TILs in vivo. Up until now, we have been blind to the behavior of cells after infusion into patients. Importantly, TIL trafficking, as well as cell survival, may be predictive of responders versus non-responders to treatment. Imaging could provide real-time surrogate markers to gauge TIL tumor homing capacity and TIL survival in each patient, which could better inform therapeutic design, as well as post-trial data analysis. The proposal has three Specific Aims: AIM 1: PFC labeling for TILs. (a) We will develop tissue culture protocols for PFC labeling of clinical TIL batches at clinical scale (>1109 cells). (b) We will rigorously evaluate the degree to which PFC labeling induces potential alterations in TIL viability and phenotype in vitro. AIM 2: In vivo rodent studies to evaluate biodistribution of TILs. Using a human patient-derived xenograft (PDX) tumor model for HNC, we will evaluate the tumor homing ability and overall biodistribution of CS-1000 labeled TILs (CS-TILs) with and without co-administration of PD-1 blockade. We will test the hypothesis that PD-1 co- administration results in increased tumor homing and accumulation. AIM 3: Clinical CS-TILs in HNC patients. In two clinical trial HNC patient cohorts, we will evaluate the feasibility of using MRI to detect (A) conventionally administered CS-TILs and (B) CS-TILs administered in combination with PD-1 blockade. The 19F MRI will be used to assay putative CS-TIL tumor homing and survival in a longitudinal fashion. Overall, this study will help expand the use of 19F MRI to a wide range of clinical trials involving T cells and other types of leukocytes.